Method for using heat-resistant mismatch endonuclease

ABSTRACT

Provided are a mismatch-specific cleavage reaction using a novel heat-resistant mismatch nuclease, a method for removing errors in a nucleic acid amplification reaction using the mismatch nuclease, a method for inhibiting the amplification of a nucleic acid having a specific base sequence during a nucleic acid amplification reaction, and a method for detecting a nucleic acid having a single-base polymorphic mutation using this inhibition method.

TECHNICAL FIELD

The present invention relates to a heat-resistant mismatch endonucleasewhich recognizes and cleaves a mismatched base pair in a double-strandednucleic acid, a composition comprising the mismatch endonuclease, and amethod of using the mismatch endonuclease.

BACKGROUND ART

In recent years, biotechnology has been remarkably developed.Particularly, in consequence of large-scale genomic analysesaccompanying advances in genomic analysis techniques, enormousinformation of genome sequences has been accumulated. In addition, basedon combinations of the above-mentioned information with analyses ofvarious physiological functions, many functional genetic mutations havebeen found. Analyses of these mutations have been used for geneticdiagnoses of human beings as well as improvement of agricultural cropsand isolation or creation of useful microorganisms, and thus havegreatly contributed to general living.

The mutation analyses are performed by direct analyses of genomicsequences or by use of enzymes that recognize mismatched base pairs. Amutation analysis method comprises detection with a factor capable ofbinding specifically to a mismatched base pair formed from a mutant-typeDNA and a wild-type DNA. A representative example of the mutationanalysis method includes detection of mutation sites by use of MutS,MutT, and MutL complexes from Escherichia coli (Patent Literature 1).

A mutation analysis method comprising use of a mismatch endonucleasewhich specifically cleaves mismatch sites is also known. In the method,a mismatch endonuclease is used to cleave a DNA in the vicinity of amismatched base pair, and the DNA fragments thus obtained are analyzedto detect the presence or absence and the position of mutations. As arepresentative example, a method comprising use of a Cell gene productfrom celery is known (Patent Literature 2), and the method is actuallyused for analyses of base mutations. However, the enzyme is notheat-resistant, and therefore cannot be used in techniques involving ahigh-temperature reaction process, such as PCR. Thus, in order to detectbase mutations, the method requires four steps of amplification,formation of mismatches, cleavage of mismatches, and detection.

In addition to mutation analyses, examples of biotechnologicaltechniques that have a lot of influence include nucleic acidamplification techniques.

A representative example of the nucleic acid amplification techniques ispolymerase chain reaction (PCR) PCR is a technique for easily amplifyinga desired fragment of a nucleic acid in vitro. PCR is an experimentaltechnique which is essential in broad fields including the fields ofbiology, medicine, and agriculture, as well as research regarding genes.PCR is also applied to detection of mutated genes and analysis ofmethylation of DNA.

Isothermal nucleic acid amplification methods such as a LAMP method andan ICAN method do not require special equipment, and therefore they areused as cheaper methods for detection of nucleic acids.

For structural analyses of the whole genome which have been performed inrecent years, a whole-genome amplification method is an importanttechnique, in particular for analyses of scarce samples.

In these nucleic acid amplification methods, incorporation of incorrectbases occurs with a constant probability. The probability has beenreduced through improvement of a polymerase or the like. However, theincorporation of incorrect bases still disturbs precise analyses.

The nucleic acid amplification techniques are used not only foramplification of a DNA having a specific nucleotide sequence but alsofor amplification of a mixture of DNAs having a common nucleotidesequence region at both ends. Specific examples of such nucleic acidamplification techniques include construction of genomic libraries orcDNA libraries. In constructing such libraries, however, a DNA moleculewith a higher content is preferentially amplified, which may disturbanalyses or screening of various kinds of DNAs.

To solve the above problem, the proportion of a DNA with a highercontent is reduced by normalization utilizing self-hybridization(Non-patent Literature 1). SSH-PCR in which PCR and self-hybridizationare combined is also used (Non-patent Literature 2). Using thesemethods, however, DNAs homologous to the DNA with a higher content maybe also removed.

In detection of a DNA by a nucleic acid amplification method, a targetDNA and a non-target DNA may compete for amplification. In other words,when a non-target DNA is amplified simultaneously with amplification ofa target DNA, it is difficult to detect the target DNA. The aboveproblem may be solved by use of real-time PCR in which probes such ascycling probes or TaqMan probes are used to detect only a target DNA. Inthe case where a non-target DNA exists in an excessively large amountrelative to a target DNA, however, it is difficult to detect the targetDNA because of false-positive reaction with many similar DNAs.

Such a problem may occur in detection of a small number of mutantalleles in the presence of normal alleles (for example, detection ofcirculating tumor genes), detection of a small number of methylated ornon-methylated alleles by epigenetic assay, detection of a small amountof fetal DNA sequences circulating in the mother's blood, and the like.

To solve the above problem, a method termed restrictionendonuclease-mediated selective polymerase chain reaction (REMS PCR) hasbeen developed (Non-patent Literature 3). This method involves use of aheat-resistant restriction enzyme. In this method, a DNA having a mutantnucleotide sequence is selectively detected using primers which, forexample, are designed so that cleavage by the restriction enzyme occursonly when a template has a normal nucleotide sequence. Depending on atarget DNA to be detected, however, there may be no heat-resistantrestriction enzyme having a recognition sequence suitable to detectionby REMS PCR.

CITATION LIST Patent Literature

-   Patent Literature 1: U.S. Pat. No. 5,922,539 B-   Patent Literature 2: WO 01/062974

Non Patent Literature

-   Non-Patent Literature 1: “Nucleic Acids Research”, 2004 February,    vol. 32, No. 3, e37-   Non-Patent Literature 2: “Methods in Molecular Biology”, 2009, vol.    496, No. 2, pp. 223-243-   Non-Patent Literature 3: “American Society for Investigative    Pathology”, 1998 August, vol. 153, No. 2, pp. 373-379

SUMMARY OF INVENTION Technical Problems

Objectives of the present invention include provision of aheat-resistant mismatch endonuclease, a composition comprising themismatch endonuclease, and a method of using the mismatch endonuclease.

Solution to Problems

As a result of intensive efforts under the above circumstances, thepresent inventors have found that a protein from archaebacteria, whichhas been regarded as a factor in the replication mechanism, has aheat-resistant mismatch endonuclease activity.

The present inventors also have found that in a nucleic acidamplification reaction in the presence of the mismatch endonuclease andan oligodeoxynucleotide which is designed to generate one or moremismatches when the oligodeoxynucleotide is hybridized with a nucleicacid having a specific nucleotide sequence, amplification of the DNAhaving the specific nucleotide sequence is inhibited.

Furthermore, the present inventors have successfully created a mutant ofthe heat-resistant mismatch endonuclease which has increasedspecificity. It has been found that when the mutant mismatchendonuclease is used, cleavage of base pairs other than a specificmismatched base pair is inhibited, allowing more specific inhibition ofamplification. Thus the present invention has been completed.

Specifically, the first aspect of the present invention provides:

A method of cleaving a double-stranded nucleic acid, the methodcomprising:

treating a double-stranded nucleic acid having a mismatched base pairwith at least one polypeptide selected from the group consisting of thefollowing (i) to (iii) to cleave both strands of the double-strandednucleic acid at the position of the mismatched base pair,

(i) a polypeptide having an amino acid sequence of SEQ ID NO:1;

(ii) a polypeptide having an amino acid sequence which differs from theamino acid sequence of SEQ ID NO:1 by substitution, deletion, insertionand/or addition of 1 to 10 amino acid residues, and having a mismatchendonuclease activity; and

(iii) a polypeptide having an amino acid sequence which shares at least50% amino acid sequence identity with the amino acid sequence of SEQ IDNO:1, and having a mismatch endonuclease activity;

The method of cleaving a double-stranded nucleic acid, wherein themismatched base pair comprises contiguous 1 to 8 mismatched base pairsexisting between two base pairs which are normally paired on thedouble-stranded nucleic acid;

A composition comprising the following (a) to (c):

-   -   (a) a DNA polymerase;    -   (b) at least one pair of oligonucleotide primers; and    -   (c) at least one polypeptide selected from the group consisting        of the following (i) to (iii):    -   (i) a polypeptide having an amino acid sequence of SEQ ID NO:1;    -   (ii) a polypeptide having an amino acid sequence which differs        from the amino acid sequence of SEQ ID NO:1 by substitution,        deletion, insertion and/or addition of 1 to 10 amino acid        residues, and having a mismatch endonuclease activity; and    -   (iii) a polypeptide having an amino acid sequence which shares        at least 50% amino acid sequence identity with the amino acid        sequence of SEQ ID NO:1, and having a mismatch endonuclease        activity;

A method of amplifying a nucleic acid, the method comprising thefollowing steps (a) and (b):

-   -   (a) preparing a composition comprising the composition according        to claim 3 and a nucleic acid molecule as a template; and    -   (b) reacting the composition obtained by step (a) under suitable        conditions to perform nucleic acid amplification;

The method of amplifying a nucleic acid, wherein the nucleicamplification is performed by a polymerase chain reaction (PCR) method,an isothermal nucleic acid amplification method, or a multipledisplacement amplification (MDA) method; A polypeptide selected from thegroup consisting of the following (A) to (C):

-   -   (A) a polypeptide having an amino acid sequence which differs        from an amino acid sequence of SEQ ID NO:1 by substitution of        tryptophan at position 77 with another amino acid residue, and        having a mismatch endonuclease activity;    -   (B) a polypeptide having an amino acid sequence which differs        from the amino acid sequence of the polypeptide of (A) by        substitution, deletion, insertion and/or addition of 1 to 10        amino acid residues other than the amino acid residue at        position 77, and having a mismatch endonuclease activity; and    -   (C) a polypeptide having an amino acid sequence which shares at        least 50% amino acid sequence identity with the amino acid        sequence of SEQ ID NO:1, in which an amino acid residue        corresponding to tryptophan at position 77 in the amino acid        sequence of SEQ ID NO:1 is substituted with another amino acid        residue, and having a mismatch endonuclease activity;

The polypeptide may be selected from the group consisting of thefollowing (A) to (C):

-   -   (A) a polypeptide having an amino acid sequence of SEQ ID NO:2;    -   (B) a polypeptide having an amino acid sequence which differs        from the amino acid sequence of SEQ ID NO:2 by substitution,        deletion, insertion and/or addition of 1 to 10 amino acid        residues other than phenylalanine at position 77, and having a        mismatch endonuclease activity; and    -   (C) a polypeptide having an amino acid sequence which shares at        least 50% amino acid sequence identity with the amino acid        sequence of SEQ ID NO:1, in which an amino acid residue        corresponding to tryptophan at position 77 in the amino acid        sequence of SEQ ID NO:1 is substituted with phenylalanine, and        having a mismatch endonuclease activity;

A method of inhibiting amplification of a nucleic acid having a specificnucleotide sequence in a nucleic acid amplification reaction, the methodcomprising a step of performing the nucleic acid amplification reactionin the presence of the following (a) to (d):

-   -   (a) an oligodeoxynucleotide which is designed to generate one to        several mismatches when the oligodeoxynucleotide is hybridized        with the nucleic acid having a specific nucleotide sequence or a        complementary strand thereof;    -   (b) a DNA polymerase;    -   (c) at least one pair of oligonucleotide primers; and    -   (d) a polypeptide having a mismatch endonuclease activity;

The method of inhibiting amplification of a nucleic acid having aspecific nucleotide sequence in a nucleic acid amplification reaction,wherein the nucleic acid amplification reaction is a polymerase chainreaction (PCR) method or an isothermal nucleic acid amplificationmethod;

The method of inhibiting amplification of a nucleic acid having aspecific nucleotide sequence in a nucleic acid amplification reaction,wherein the polypeptide having a mismatch endonuclease activity is atleast one polypeptide selected from the group consisting of thefollowing (i) to (vi):

-   -   (i) a polypeptide having an amino acid sequence of SEQ ID NO:1;    -   (ii) a polypeptide having an amino acid sequence which differs        from the amino acid sequence of SEQ ID NO:1 by substitution,        deletion, insertion and/or addition of 1 to 10 amino acid        residues, and having a mismatch endonuclease activity;    -   (iii) a polypeptide having an amino acid sequence which shares        at least 50% amino acid sequence identity with the amino acid        sequence of SEQ ID NO:1, and having a mismatch endonuclease        activity;    -   (iv) a polypeptide having an amino acid sequence of SEQ ID NO:2;    -   (v) a polypeptide having an amino acid sequence which differs        from the amino acid sequence of SEQ ID NO:2 by substitution,        deletion, insertion and/or addition of 1 to 10 amino acid        residues other than phenylalanine at position 77, and having a        mismatch endonuclease activity; and    -   (vi) a polypeptide having an amino acid sequence which shares at        least 50% amino acid sequence identity with the amino acid        sequence of SEQ ID NO:1, in which an amino acid residue        corresponding to tryptophan at position 77 in the amino acid        sequence of SEQ ID NO:1 is substituted with phenylalanine, and        having a mismatch endonuclease activity;

A method of preferentially amplifying a target DNA, the methodcomprising inhibiting amplification of a DNA having a nucleotidesequence different from that of the target DNA in one to severalnucleotides by the method of inhibiting amplification of a nucleic acidhaving a specific nucleotide sequence in a nucleic acid amplificationreaction:

The method of preferentially amplifying a target DNA, which is used foramplifying a DNA having a wild-type nucleotide sequence or the DNAcontaining a single nucleotide polymorphism mutation, wherein theamplification of the wild-type DNA and the amplification of the DNAcontaining a single nucleotide polymorphism mutation are distinguishedfrom each other; The method of preferentially amplifying a target DNA,wherein the single nucleotide polymorphism mutation correlates withcanceration, or a therapeutic effect of an agent for the treatment of acancer; and

The method of preferentially amplifying a target DNA, wherein the DNAamplification reaction is performed by a polymerase chain reaction (PCR)method or an isothermal nucleic acid amplification method.

Effects of the Invention

According to the present invention, a heat-resistant mismatchendonuclease which has great utility in biotechnology, a compositioncomprising the mismatch endonuclease, and a method of using the mismatchendonuclease are provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of polyacrylamide gel electrophoresis in Example 2.

FIG. 2 shows mismatch cleavage activities of protein PF0012 on the basisof increases of fluorescent intensity in Example 2.

FIG. 3 shows mismatch cleavage activities of PF0012 homologous proteinon the basis of increases of fluorescent intensity in Example 3.

FIG. 4 shows mismatch cleavage activities of mutant PF0012 homologousprotein on the basis of increases of fluorescent intensity in Example 5.

FIG. 5 shows inhibition of amplification of a DNA having a specificnucleotide sequence by real-time PCR technique in Example 6.

FIG. 6 shows detection of a single nucleotide-mutated gene by an HRManalysis method.

DESCRIPTION OF EMBODIMENTS

In the present invention, the word “mismatch” refers to base pairingsdifferent from Watson-Crick base pairs present in double-strandednucleic acids, in other words, binding of bases in combinations otherthan base pairings of G (guanine base)-C (cytosine base), and A (adeninebase)-T (thymine base) or U (uracil base).

As used herein, a polypeptide having a mismatch endonuclease activity(sometimes, referred to as a mismatch endonuclease) means a nucleasehaving the activity of cleaving mismatch sites present indouble-stranded nucleic acids. The mismatch endonuclease activityincludes an activity of cleaving phosphodiester bonds adjacent tonucleotides forming mismatched base pairs, and an activity of cleavingphosphodiester bonds adjacent to nucleotides located 1 to 5, preferably1 to 3 base pairs away from mismatched base pairs. In the presentinvention, the mismatch endonuclease may be a nuclease having theactivity of specifically recognizing a specific mismatched base pair ina double-stranded nucleic acid to cleave the double-stranded nucleicacid (for example, a nuclease which specifically recognizes GTmismatches, or a nuclease which specifically recognizes GT mismatchesand GG mismatches). In the present invention, the heat-resistantmismatch endonuclease may have, in addition to the activity of cleavingmismatch sites present in double-stranded nucleic acids, the activity ofrecognizing single-stranded DNAs, junction parts between single-strandednucleic acids and double-stranded nucleic acids, double flap structure,replication fork structure, D-loop structure, and/or Holiday junctionstructure with a nick to cleave the nucleic acids. As used herein, theheat-resistant mismatch endonuclease means a nuclease that exhibits theactivity of cleaving mismatch sites in double-stranded nucleic acids attemperature of 50° C. or higher. In the present invention, aheat-resistant mismatch endonuclease is preferably used.

Examples of the heat-resistant mismatch endonuclease used in the presentinvention include, but not limited to, polypeptides having aheat-resistant mismatch endonuclease activity from archaebacteria. Thepresent inventors have found that polypeptide PF0012 (RefSeq ID: NP577741, SEQ ID NO:1) from Pyrococcus furiosus, which has been to dateregarded as a factor in the replication mechanism, is a heat-resistantmismatch endonuclease. Furthermore, the present inventors have foundthat polypeptide MJ_0225 (RefSeq ID: NP_247194, SEQ ID NO:8) fromMethanocaldococcus jannaschii and polypeptide TERMP_01877 (RefSeq ID:YP_004072075, SEQ ID NO:7) from Thermococcus barophilus, which arehomologs of PF0012, are also heat-resistant mismatch endonucleases.MJ_0225 and TERMP_01877 have 57% and 73% sequence identity with SEQ IDNO:1 respectively. Preferred examples of the heat-resistant mismatchendonucleases used in the present invention include a polypeptide havingan amino acid sequence of SEQ ID NO:1; a polypeptide having an aminoacid sequence which differs from the amino acid sequence of SEQ ID NO:1by substitution, deletion, insertion and/or addition of 1 to 10 aminoacid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues; and a polypeptide having an amino acid sequence which sharesat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, orat least 75% amino acid sequence identity, preferably at least 80% aminoacid sequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:1. Preferred examples of theheat-resistant mismatch endonucleases used in the present invention alsoinclude a polypeptide having an amino acid sequence of SEQ ID NO:7; apolypeptide having an amino acid sequence which differs from the aminoacid sequence of SEQ ID NO:7 by substitution, deletion, insertion and/oraddition of 1 to 10 amino acid residues, for example 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid residues; and a polypeptide having an aminoacid sequence which shares at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, or at least 75% amino acid sequence identity,preferably at least 80% amino acid sequence identity, more preferably atleast 85% amino acid sequence identity, still more preferably at least90% amino acid sequence identity, most preferably at least 95% aminoacid sequence identity with the amino acid sequence of SEQ ID NO:7.Preferred examples of the heat-resistant mismatch endonucleases used inthe present invention also include a polypeptide having an amino acidsequence of SEQ ID NO:8; a polypeptide having an amino acid sequencewhich differs from the amino acid sequence of SEQ ID NO:8 bysubstitution, deletion, insertion and/or addition of 1 to 10 amino acidresidues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues; and a polypeptide having an amino acid sequence which sharesat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, orat least 75% amino acid sequence identity, preferably at least 80% aminoacid sequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:8.

Furthermore, the present inventors have found that a polypeptide havingan amino acid sequence which differs from the amino acid sequence of SEQID NO:1 by substitution of tryptophan at position 77 with another aminoacid residue, preferably phenylalanine is a heat-resistant mismatchendonuclease that specifically recognizes mismatch G (guanine base)-G(guanine base). As a result of measurement, the cleavage activity of themismatch endonuclease on the mismatched base pair formed from guaninebases was 10 times or more the cleavage activity on other mismatchedbase pairs. Thus, an aspect of the present invention includes apolypeptide having an amino acid sequence of SEQ ID NO:2 thus createdand homologs thereof. Examples of the polypeptide having an amino acidsequence of SEQ ID NO:2 and homologs thereof include a polypeptidehaving an amino acid sequence which differs from the amino acid sequenceof SEQ ID NO:1 by substitution of tryptophan at position 77 with anotheramino acid residue, and having a mismatch endonuclease activity; apolypeptide having an amino acid sequence which differs from the aminoacid sequence of the above-mentioned polypeptide by substitution,deletion, insertion and/or addition of 1 to 10 amino acid residues, forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues other thanthe amino acid residue at position 77, and having a mismatchendonuclease activity; and a polypeptide having an amino acid sequencewhich shares at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, or at least 75% amino acid sequence identity, preferably atleast 80% amino acid sequence identity, more preferably at least 85%amino acid sequence identity, still more preferably at least 90% aminoacid sequence identity, most preferably at least 95% amino acid sequenceidentity with the amino acid sequence of SEQ ID NO:1, in which an aminoacid residue corresponding to tryptophan at position 77 in the aminoacid sequence of SEQ ID NO:1 is an amino acid residue other thantryptophan, and having a mismatch endonuclease activity. Such mismatchendonucleases are suitable for various uses as mentioned later, forexample, a method comprising removing a DNA containing a specific DNAsequence and amplifying and detecting other DNAs.

The mismatch endonuclease activity may be measured by use of adouble-stranded nucleic acid containing a mismatched base pair as asubstrate. Specifically, after a double-stranded nucleic acid containinga mismatched base pair is prepared, the double-stranded nucleic acid isreacted with a mismatch endonuclease in which the amount ofdouble-stranded nucleic acid is excess relative to the amount of themismatch endonuclease, and then, the amount of nucleic acids cleaved perunit time is measured. The cleaved double-stranded nucleic acids can bequantified separately from non-cleaved nucleic acids, for example, byelectrophoresis. A double-stranded nucleic acid double labeled with afluorescent substance and a quencher substance may be used so that anincrease in fluorescent intensity can be detected only when thedouble-stranded nucleic acid is cleaved. Using such a double-strandednucleic acid double labeled with a fluorescent substance and a quenchersubstance, the mismatch endonuclease activity can be easily determinedby measuring the fluorescent intensity in a reaction mixture at suitabletime intervals. The cleavage activity on a specific mismatched base paircan be determined by changing the bases forming a mismatched base pairpresent in a double-stranded nucleic acid used as a substrate.

The method of cleaving a double-stranded nucleic acid of the presentinvention comprises treating a double-stranded nucleic acid having amismatched base pair with a polypeptide having the amino acid sequenceof SEQ ID NO:1 or a homolog thereof. Examples of the homolog of thepolypeptide having the amino acid sequence of SEQ ID NO:1 include, butnot limited to, a polypeptide having an amino acid sequence whichdiffers from the amino acid sequence of SEQ ID NO:1 by substitution,deletion, insertion and/or addition of 1 to 10 amino acid residues, forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, and havinga mismatch endonuclease activity; a polypeptide having an amino acidsequence which shares at least 50% amino acid sequence identity, atleast 55%, at least 60%, at least 65%, at least 70%, or at least 75%amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:1, and having a mismatchendonuclease activity; and a polypeptide having the amino acid sequenceof SEQ ID NO:2 and a homolog thereof. Examples of organisms from whichthe polypeptide used in the double-stranded nucleic acid cleavage methodof the present invention is derived include archaebacteria, preferablyheat-resistant archaebacteria, more preferably Pyrococcus furiosus,Thermococcus barophilus, and Methanocaldococcus jannaschii. In thedouble-stranded nucleic acid cleavage method of the present invention,polypeptide MJ_0225 (RefSeq ID: NP_247194, SEQ ID NO:8) fromMethanocaldococcus jannaschii, which is a homolog of PF0012, or ahomolog of MJ_0225, or polypeptide TERMP_01877 (RefSeq ID: YP_004072075,SEQ ID NO:7) from Thermococcus barophilus, which is a homolog of PF0012,or a homolog of TERMP_01877 may be also used. Examples of the homolog ofthe polypeptide having the amino acid sequence of SEQ ID NO:7 include,but not limited to, a polypeptide having an amino acid sequence whichdiffers from the amino acid sequence of SEQ ID NO:7 by substitution,deletion, insertion and/or addition of 1 to 10 amino acid residues, forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, and havinga mismatch endonuclease activity; and a polypeptide having an amino acidsequence which shares at least 50%, at least 55%, at least 60%, at least65%, at least 70%, or at least 75% amino acid sequence identity,preferably at least 80% amino acid sequence identity, more preferably atleast 85% amino acid sequence identity, still more preferably at least90% amino acid sequence identity, most preferably at least 95% aminoacid sequence identity with the amino acid sequence of SEQ ID NO:7, andhaving a mismatch endonuclease activity. Examples of the homolog of thepolypeptide having the amino acid sequence of SEQ ID NO:8 include, butnot limited to, a polypeptide having an amino acid sequence whichdiffers from the amino acid sequence of SEQ ID NO:8 by substitution,deletion, insertion and/or addition of 1 to 10 amino acid residues, forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, and havinga mismatch endonuclease activity; and a polypeptide having an amino acidsequence which shares at least 50%, at least 55%, at least 60%, at least65%, at least 70%, or at least 75% amino acid sequence identity,preferably at least 80% amino acid sequence identity, more preferably atleast 85% amino acid sequence identity, still more preferably at least90% amino acid sequence identity, most preferably at least 95% aminoacid sequence identity with the amino acid sequence of SEQ ID NO:8, andhaving a mismatch endonuclease activity.

The mismatched base pair in the double-stranded nucleic acid cleavagemethod of the present invention is present within the double-strandednucleic acid (between two base pairs normally base-pairing). Themismatched base pairs are not limited to one mismatched base pairpresent within the double-stranded nucleic acid. The mismatched basepairs also include plural mismatched base pairs present at intervalswithin the double-stranded nucleic acid, and two or more contiguousmismatched base pairs present within the double-stranded nucleic acid.Examples of the mismatched base pairs in the double-stranded nucleicacid cleavage method of the present invention include preferably 1 to 8contiguous mismatched base pairs present within the double-strandednucleic acid, more preferably 1 to 4 contiguous mismatched base pairspresent within the double-stranded nucleic acid, and still morepreferably 2 contiguous mismatched base pairs or one mismatched basepair present within the double-stranded nucleic acid. In thedouble-stranded nucleic acid cleavage method of the present invention,when plural mismatched base pairs are present within the double-strandednucleic acid, the plural mismatched base pairs may be mismatched basepairs of the same kind or different kinds.

Examples of the double-stranded nucleic acid having a mismatched basepair include a nucleic acid from a organism sample, for example, a PCRproduct, a genomic DNA, or a fragment thereof, and a synthetic nucleicacid. The double-stranded nucleic acid having a mismatched base pair mayalso be a nucleic acid mixture obtained by melting and reannealing of amixture of plural organism samples, or a mixture of nucleic acids froman organism sample and a synthetic nucleic acid. For example, when anucleic acid containing a mutation and a wild-type nucleic acid aremixed, melted, and reannealed, a mismatched base pair is formed andcleavage by a mismatch endonuclease occurs at the position of themismatched base pair. After cleavage by a mismatch endonuclease, thesize of the nucleic acid fragment thus cleaved is observed to determinethe presence or absence and the position of a mutation. Use of themismatch endonuclease of the present invention allows mutation analysisby one-step reaction in which the mismatch endonuclease is simply addedto a reaction mixture for a nucleic acid amplification method such asPCR. It is known that when the number of cycles in PCR is increased toexceed a certain number, no effect in the amplification is obtained. Themain causes are depletion of the nucleic acid added to the reactionmixture, or competition between primers and reaction products forannealing. At that time, annealing between the reaction products occurs.If a template containing a mutation and a wild-type template coexist,the reaction products amplified from these are annealed each other togenerate a mismatched base pair at the mutation site. Thus, mutationanalysis can be done simply by performing PCR in the presence of themismatch endonuclease of the present invention through more cycles thanusual. Specifically, the present invention provides a mutation analysismethod comprising treatment of a double-stranded nucleic acid with apolypeptide having the amino acid sequence of SEQ ID NO:1 or a homologthereof. The present invention further provides a mutation analysismethod comprising treatment of a double-stranded nucleic acid with apolypeptide having the amino acid sequence of SEQ ID NO:7 or a homologthereof. The present invention further provides a mutation analysismethod comprising treatment of a double-stranded nucleic acid with apolypeptide having the amino acid sequence of SEQ ID NO:8 or a homologthereof.

In the process of nucleic acid amplification reaction, thedouble-stranded nucleic acid cleavage method of the present inventioncan be performed. By addition of a mismatch endonuclease to a reactionmixture for nucleic acid amplification, a double-stranded nucleic acidhaving a mismatched base pair generated by incorporation of an incorrectbase during the amplification process is cleaved.

As a result, amplification of a nucleic acid having a different sequencefrom that of a template nucleic acid before the reaction initiation isinhibited. Thus, nucleic acid amplification with a decreased error rateis attained. Specifically, the present invention provides a nucleic acidamplification method comprising a step of cleaving a double-strandednucleic acid having a mismatched base pair by using a polypeptide havingthe amino acid sequence of SEQ ID NO:1 or a homolog thereof. The presentinvention further provides a nucleic acid amplification methodcomprising a step of cleaving a double-stranded nucleic acid having amismatched base pair by using a polypeptide having the amino acidsequence of SEQ ID NO:7 or a homolog thereof. The present inventionfurther provides a nucleic acid amplification method comprising a stepof cleaving a double-stranded nucleic acid having a mismatched base pairby using a polypeptide having the amino acid sequence of SEQ ID NO:8 ora homolog thereof. The step of cleaving a double-stranded nucleic acidhaving a mismatched base pair may be performed simultaneously with astep of nucleic acid amplification. An aspect of the present inventionalso includes a composition comprising (a) a DNA polymerase; (b) atleast one pair of oligonucleotide primers; and (c) at least onepolypeptide selected from the group consisting of (i) a polypeptidehaving an amino acid sequence of SEQ ID NO:1; (ii) a polypeptide havingan amino acid sequence which differs from the amino acid sequence of SEQID NO:1 by substitution, deletion, insertion and/or addition of 1 to 10amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid residues, and having a mismatch endonuclease activity; and (iii) apolypeptide having an amino acid sequence which shares at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, or at least 75%amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:1, and having a mismatchendonuclease activity. As another aspect of the present invention, acomposition is provided, comprising (a) a DNA polymerase; (b) at leastone pair of oligonucleotide primers; and (c) at least one polypeptideselected from the group consisting of (i) a polypeptide having an aminoacid sequence of SEQ ID NO:7; (ii) a polypeptide having an amino acidsequence which differs from the amino acid sequence of SEQ ID NO:7 bysubstitution, deletion, insertion and/or addition of 1 to 10 amino acidresidues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues, and having a mismatch endonuclease activity; and (iii) apolypeptide having an amino acid sequence which shares at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, or at least 75%amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:7, and having a mismatchendonuclease activity. As another aspect of the present invention, acomposition is also provided, comprising (a) a DNA polymerase; (b) atleast one pair of oligonucleotide primers; and (c) at least onepolypeptide selected from the group consisting of (i) a polypeptidehaving an amino acid sequence of SEQ ID NO:8; (ii) a polypeptide havingan amino acid sequence which differs from the amino acid sequence of SEQID NO:8 by substitution, deletion, insertion and/or addition of 1 to 10amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid residues, and having a mismatch endonuclease activity; and (iii) apolypeptide having an amino acid sequence which shares at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, or at least 75%amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:8, and having a mismatchendonuclease activity.

An aspect of the present invention also includes a nucleic acidamplification method comprising a step of preparing a compositioncomprising the composition for nucleic acid amplification as mentionedabove and a nucleic acid molecule as a template, and a step of reactingthe composition thus obtained under suitable conditions to performnucleic acid amplification.

The above-mentioned composition may further contain at least oneselected from the group consisting of a reaction buffer, a divalentmetal ion, a deoxyribonucleotide, an oligonucleotide probe, and anintercalating dye. When the above-mentioned composition is used innucleic acid amplification reaction, the composition may further containa nucleic acid as a template for the nucleic acid amplificationreaction. The reaction buffer means a compound or mixture having theactivity of decreasing variation of hydrogen-ion concentration (pH). Amixed solution of weak acid and a salt thereof or weak base and a saltthereof is broadly used as the reaction buffer for the purpose of pHcontrol, because the mixed solution has strong buffering action.Examples of the reaction buffer used in the present invention includeGood's buffers such as Tris-HCl, and HEPES-KOH, and phosphate bufferssuch as a sodium phosphate buffer. Examples of the divalent metal ioninclude a magnesium ion, a manganese ion, a zinc ion, and a cobalt ion.The divalent metal ion may be provided as a salt form such as achloride, a sulfate, or an acetate.

Examples of a method for the nucleic acid amplification include, but notlimited to, a method of amplifying a DNA. Examples of the method ofamplifying a DNA include a polymerase chain reaction (PCR) method, amultiple displacement amplification (MDA) method, and an isothermalnucleic acid amplification method such as an ICAN method and a LAMPmethod.

The concentration of a polypeptide having a mismatch endonucleaseactivity in the above-mentioned composition for nucleic acidamplification reaction may be determined by determining a concentrationthat does not inhibit a DNA amplification reaction or a concentrationeffective for cleavage of a mismatched based pair in each reactionsystem as appropriate.

As the at least one pair of primers contained in the above-mentionedcomposition for nucleic acid amplification reaction, two or more primerssuitable for each nucleic acid amplification method are selected. Theseprimers may be DNA primers, RNA primers, or chimeric primers in which apart of a DNA molecule is replaced by RNA, as long as the desiredamplification is attained. The primers may also be primers containing aknown nucleic acid analog, and labeled primers, for example, with afluorescent dye for the purpose of detection.

The present inventors have further found that amplification of a nucleicacid having a specific nucleotide sequence in a nucleic acidamplification reaction can be inhibited by use of a mismatchendonuclease and a suitably designed oligodeoxynucleotide. Thus anaspect of the present invention also includes a method of inhibitingamplification of a nucleic acid having a specific nucleotide sequence ina nucleic acid amplification reaction, comprising a step of performingthe nucleic acid amplification reaction in the presence of (a) anoligodeoxynucleotide which is designed to generate one to severalmismatches when the oligodeoxynucleotide is hybridized with the nucleicacid having a specific nucleotide sequence, (b) a DNA polymerase, (c) atleast one pair of oligonucleotide primers, and (d) a polypeptide havinga mismatch endonuclease activity. An aspect of the present inventionalso includes a method of preferentially amplifying a target DNA,comprising inhibiting amplification of a DNA having a specificnucleotide sequence different from the nucleotide sequence of the targetDNA in one to several nucleotides by use of the above-mentioned methodof inhibiting amplification of a nucleic acid having a specificnucleotide sequence in a nucleic acid amplification reaction.

The oligodeoxynucleotide in the above (a) is not particularly limited,as long as it is an oligodeoxynucleotide designed to generate one toseveral mismatches when it is hybridized with a nucleic acid having aspecific nucleotide sequence. The oligodeoxynucleotide may be aso-called chimeric oligodeoxynucleotide in which a part of a DNAmolecule is replaced by RNA. The 3′ end of the oligodeoxynucleotide maybe modified so as to inhibit an extension reaction from theoligodeoxynucleotide by a DNA polymerase, to which the present inventionis not particularly limited. Examples of the modification includeamination. The oligodeoxynucleotide may be protected from cleavage witha deoxyribonuclease by phosphorothioation or other modifications, aslong as the nucleic acid to which the oligodeoxynucleotide is boundundergoes cleavage with the polypeptide having a mismatch endonucleaseactivity. The oligodeoxynucleotide may be labeled with a fluorescent dyeor a quencher for the purpose of detection.

The length of the oligodeoxynucleotide may be appropriately determinedso that the oligodeoxynucleotide can be hybridized with the nucleic acidhaving a specific nucleotide sequence under conditions of the reactionperformed. The position of a mismatch generated when theoligodeoxynucleotide is hybridized with the nucleic acid having aspecific nucleotide sequence is preferably at least 3 nucleotides awayfrom the 5′ end and 3′ end of the oligodeoxynucleotide.

For the method of inhibiting amplification of a nucleic acid having aspecific nucleotide sequence in a nucleic acid amplification reaction ofthe present invention, any mismatch endonuclease having the activity ofspecifically cleaving a mismatch site can be used. For example, when aheat-resistant DNA polymerase is used in a nucleic acid amplificationmethod including a reaction at a high temperature such as a PCR method,a heat-resistant mismatch endonuclease is preferably used. In such acase, preferably used is the heat-resistant mismatch endonuclease, thatis, at least one polypeptide selected from the group consisting of (i) apolypeptide having an amino acid sequence of SEQ ID NO:1; (ii) apolypeptide having an amino acid sequence which differs from the aminoacid sequence of SEQ ID NO:1 by substitution, deletion, insertion and/oraddition of 1 to 10 amino acid residues, for example 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid residues, and having a mismatch endonucleaseactivity; and (iii) a polypeptide having an amino acid sequence whichshares at least 50%, at least 55%, at least 60%, at least 65%, at least70%, or at least 75% amino acid sequence identity, preferably at least80% amino acid sequence identity, more preferably at least 85% aminoacid sequence identity, still more preferably at least 90% amino acidsequence identity, most preferably at least 95% amino acid sequenceidentity with the amino acid sequence of SEQ ID NO:1, and having amismatch endonuclease activity; (iv) a polypeptide having an amino acidsequence of SEQ ID NO:7; (v) a polypeptide having an amino acid sequencewhich differs from the amino acid sequence of SEQ ID NO:7 bysubstitution, deletion, insertion and/or addition of 1 to 10 amino acidresidues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues, and having a mismatch endonuclease activity; and (vi) apolypeptide having an amino acid sequence which shares at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, or at least 75%amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:7, and having a mismatchendonuclease activity; (vii) a polypeptide having an amino acid sequenceof SEQ ID NO:8; (viii) a polypeptide having an amino acid sequence whichdiffers from the amino acid sequence of SEQ ID NO:8 by substitution,deletion, insertion and/or addition of 1 to 10 amino acid residues, forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, and havinga mismatch endonuclease activity; and (ix) a polypeptide having an aminoacid sequence which shares at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, or at least 75% amino acid sequence identity,preferably at least 80% amino acid sequence identity, more preferably atleast 85% amino acid sequence identity, still more preferably at least90% amino acid sequence identity, most preferably at least 95% aminoacid sequence identity with the amino acid sequence of SEQ ID NO:8, andhaving a mismatch endonuclease activity, but to which the presentinvention is not particularly limited.

For the method of inhibiting amplification of a nucleic acid having aspecific nucleotide sequence in a nucleic acid amplification reaction ofthe present invention, a mismatch endonuclease having the activity ofspecifically cleaving only a double-stranded nucleic acid containing aspecific mismatched base pair is preferably used. In such a case, thenucleotide sequence whose amplification is inhibited can be limited toone kind of nucleotide sequence. For example, a polypeptide having theamino acid sequence of SEQ ID NO:2 which differs from the amino acidsequence of SEQ ID NO:1 by substitution of tryptophan at position 77with another amino acid residue specifically cleaves a double-strandednucleic acid containing a G (guanine base)-G (guanine base) mismatch.Thus, when the polypeptide having the amino acid sequence of SEQ ID NO:2is used, a double-stranded nucleic acid in which a base other than G(guanine base) is present at the mismatch site is not cleaved and doesnot undergo inhibition of amplification. Specifically, a polypeptidehaving an amino acid sequence which differs from the amino acid sequenceof SEQ ID NO:1 by substitution of tryptophan at position 77 with anotheramino acid residue, and a homolog thereof are preferably used for themethod of inhibiting amplification of a nucleic acid having a specificnucleotide sequence.

Therefore, the present invention further provides a composition fornucleic acid amplification reaction, comprising the following (a) to(d):

(a) an oligodeoxynucleotide which is designed to generate one to severalmismatches when the oligodeoxynucleotide is hybridized with a nucleicacid having a specific nucleotide sequence or a complementary strandthereof;

(b) a DNA polymerase;

(c) at least one pair of oligonucleotide primers; and

(d) at least one polypeptide selected from the group consisting of thefollowing (i) to (xii):

(i) a polypeptide having an amino acid sequence of SEQ ID NO:1;

(ii) a polypeptide having an amino acid sequence which differs from theamino acid sequence of SEQ ID NO:1 by substitution, deletion, insertionand/or addition of 1 to 10 amino acid residues, for example 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acid residues, and having a mismatchendonuclease activity;

(iii) a polypeptide having an amino acid sequence which shares at least50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least75% amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:1, and having a mismatchendonuclease activity;

(iv) a polypeptide having an amino acid sequence of SEQ ID NO:2;

(v) a polypeptide having an amino acid sequence which differs from theamino acid sequence of SEQ ID NO:2 by substitution, deletion, insertionand/or addition of 1 to 10 amino acid residues, for example 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acid residues other than phenylalanine atposition 77, and having a mismatch endonuclease activity;

(vi) a polypeptide having an amino acid sequence which shares at least50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least75% amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:1, in which an amino acid residuecorresponding to tryptophan at position 77 in the amino acid sequence ofSEQ ID NO:1 is substituted with another amino acid residue, and having amismatch endonuclease activity;

(vii) a polypeptide having an amino acid sequence of SEQ ID NO:7;

(viii) a polypeptide having an amino acid sequence which differs fromthe amino acid sequence of SEQ ID NO:7 by substitution, deletion,insertion and/or addition of 1 to 10 amino acid residues, for example 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, and having a mismatchendonuclease activity;

(ix) a polypeptide having an amino acid sequence which shares at least50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least75% amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:7, and having a mismatchendonuclease activity;

(x) a polypeptide having an amino acid sequence of SEQ ID NO:8;

(xi) a polypeptide having an amino acid sequence which differs from theamino acid sequence of SEQ ID NO:8 by substitution, deletion, insertionand/or addition of 1 to 10 amino acid residues, for example 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acid residues, and having a mismatchendonuclease activity; and

(xii) a polypeptide having an amino acid sequence which shares at least50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least75% amino acid sequence identity, preferably at least 80% amino acidsequence identity, more preferably at least 85% amino acid sequenceidentity, still more preferably at least 90% amino acid sequenceidentity, most preferably at least 95% amino acid sequence identity withthe amino acid sequence of SEQ ID NO:8, and having a mismatchendonuclease activity. The above-mentioned composition may furthercontain at least one selected from the group consisting of a reactionbuffer, a divalent metal ion, a deoxyribonucleotide, an oligonucleotideprobe, and an intercalating dye. The above-mentioned composition mayfurther contain a nucleic acid as a template for nucleic amplificationreaction.

The method of inhibiting amplification of a nucleic acid having aspecific nucleotide sequence in a nucleic acid amplification reaction ofthe present invention can be performed by any nucleic acid amplificationmethods. A method of amplifying a DNA is preferably used, but which thepresent invention is limited to. The present invention can be performed,for example, by a PCR method, an MDA method, or an isothermal nucleicacid amplification method such as a LAMP method or an ICAN method.

The method of inhibiting amplification of a nucleic acid having aspecific nucleotide sequence in a nucleic acid amplification reaction ofthe present invention can be applied to amplification of any nucleicacids. When a DNA is used as a target to be amplified, examples of theDNA include a DNA present in an artificially prepared DNA mixture, asample from environment, an organism sample, or a DNA mixture preparedfrom the above-mentioned sample. Examples of the organism sampleinclude, but not limited to, samples from mammals such as human.Examples of the DNA mixture include, but not limited to, a mixture ofgenomic DNA fragments, a mixture of cDNAs generated from mRNAs byreverse transcription reaction, and a mixture of plural PCR products.Examples of the DNA having a specific nucleotide sequence which issubjected to inhibition of amplification include a reverse transcriptionproduct from a rRNA which is not separated and remains, and alow-molecular DNA which is generated by pairing between primers. When agene library followed by functional screening is amplified, a librarycapable of efficiently searching an unknown gene can be made byinhibiting amplification of a DNA having a sequence of a known geneexhibiting a positive signal.

The concentration of the polypeptide having a mismatch endonucleaseactivity in (d) of the method of inhibiting amplification of a nucleicacid having a specific nucleotide sequence in a nucleic acidamplification reaction may be determined by examining a concentrationthat does not inhibit DNA amplification reaction or a concentrationeffective for cleavage of a mismatched based pair in each reactionsystem as appropriate. The concentration of the oligodeoxynucleotide in(a) may be determined by optimizing the usage concentration whileconsidering the amount of a template or amplification efficiency of thetarget DNA. For example, the concentration of the oligodeoxynucleotidecan be 0.1 to 10 times the concentration of a primer used foramplification reaction.

The method of preferentially amplifying a target DNA may furthercomprise a step of detecting the amplified target DNA. This aspect ofthe present invention, as used herein, is sometimes referred to as “thedetection method of the present invention”. For example, according tothe detection method of the present invention in which a DNA is used asa target to be detected, when a DNA that is not a target to be detected(a DNA having a specific nucleotide sequence) exists in an excessivelylarge amount relative to a DNA that is a target to be detected (a targetDNA), amplification of the non-target DNA as a template is inhibited byvirtue of the oligodeoxynucleotide in (a) and the polypeptide having amismatch endonuclease activity in (d) of the method of inhibitingamplification of a nucleic acid having a specific nucleotide sequence ina nucleic acid amplification reaction, and therefore the target DNA tobe detected can be detected.

The detection method of the present invention enables to distinctivelydetect the wild-type and the mutant-type, for example, of a nucleic acidcorresponding to a gene wherein a mutaion in the gene is known to bepresent. When the detection method of the present invention is performedusing a DNA having a wild-type nucleotide sequence as the nucleic acidhaving a specific nucleotide sequence, a small number of a mutant allelecan be detected in the presence of an excessively large amount of thenormal allele (i.e., a DNA having the wild-type nucleotide sequence).For example, the method of the present invention is useful for detectionof a circulating tumor DNA, or detection of a small amount of a fetalDNA sequence contained in the mother's blood. Examples of the mutationinclude microdeletion and point mutation. Polymorphisms generated bypoint mutation are called single nucleotide polymorphisms (SNPs). Asused herein, a DNA having a mutant nucleotide sequence among SNPs issometimes referred to as a DNA having a single nucleotide polymorphismmutation.

The nucleic acid having a specific nucleotide sequence may be preferablya nucleic acid containing at least one single nucleotide polymorphismselected from the group consisting of a single nucleotide polymorphismused as a tumor marker, a single nucleotide polymorphism correlatingwith a therapeutic effect of an agent for the treatment of cancer, and asingle nucleotide polymorphism known to correlate with canceration ofcells, but which the present invention is not particularly limited to.Examples of SNPs include those frequently found in tumor cells, andthose known to correlate with a therapeutic effect of an agent for thetreatment of cancer or carcinogenesis. Examples of such SNPs includeSNPs of K-ras genes, B-raf genes, and epidermal growth factor receptor(EGFR) genes. Somatic mutations in the K-ras gene are frequently foundin colorectal cancer, lung adenocarcinoma, thyroid cancer, and the like.Somatic mutations in the B-raf gene are frequently found in colorectalcancer, malignant melanoma, papillary thyroid cancer, non-small celllung cancer, lung adenocarcinoma, and the like. Somatic mutations in theEGFR gene are frequently found in various solid tumors. It is known thatthe treatment of a cancer with an EGFR inhibitor such as gefitinib orerlotinib is likely to be effective when the EGFR gene in the cancertissue has a specific single nucleotide polymorphism mutation. Incontrast, it is known that a cancer is likely to be resistant to an EGFRinhibitor when the K-ras gene in the cancer tissue has a singlenucleotide polymorphism mutation.

The detection method of the present invention may be performed using, asthe material, a DNA obtained after treatment of a composition containinga methylated DNA extracted from an organism sample with bisulfite.According to the detection method of the present invention, detection ofa small number of a methylated allele in the presence of an excessivelylarge amount of a non-methylated allele, or detection of a small numberof a non-methylated allele in the presence of an excessively largeamount of a methylated allele can be performed.

As the treatment with bisulfite, a known bisulfite method which is usedfor detection of a methylated DNA can be used. By the treatment,non-methylated cytosine is changed into uracil, whereas methylatedcytosine is not changed. When a reaction mixture treated with bisulfiteis subjected to amplification by PCR, uracil is changed into thymine andmethylated cytosine is changed into cytosine. In other words, detectionof a small number of a methylated allele in the presence of anexcessively large amount of a non-methylated allele at a specific site,and detection of a small number of a non-methylated allele in thepresence of an excessively large amount of a methylated allelerespectively correspond to examination of the presence of cytosine inthe presence of an excessively large amount of thymine, and examinationof the presence of thymine in the presence of an excessively largeamount of cytosine. When amplification of an excessively large amount ofDNA containing thymine or cytosine is inhibited, the presence of a smallnumber of a methylated allele or non-methylated allele is easilyexamined.

For the step of detecting the target nucleic acid in the detectionmethod of the present invention, electrophoresis, nucleotide sequenceanalysis, or real-time PCR using a probe such as a cycling probe or aTaqMan probe can be used. For these detection methods, conventionaltechniques can be directly used. In particular, use of a high resolutionmelting (HRM) analysis method allows amplification and detection of aDNA of interest by one step, and thus rapid and simple examination ofthe DNA of interest is attained.

EXAMPLES

The present invention will be more specifically explained by way ofExamples, which the present invention is not limited to.

Preparation Example 1: Preparation of Genomic DNA

A genomic DNA of Pyrococcus furiosus was prepared following a methoddescribed in Example 1 of Japan Patent No. 3742659. Genomic DNAs ofThermococcus barophilus and Methanocaldococcus jannaschii were preparedfollowing a method described in Example 8 of WO02/22831, except thatThermococcus barophilus and Methanocaldococcus jannaschii were usedinstead of Thermococcus litoralis as a microbial strain. These microbialstrains are available from Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH.

Preparation Example 2: Preparation of Protein PF0012

(1) Preparation of pET-PF12, Plasmid for Expression of PF0012

For functional analysis of protein PF0012, a system for forcedexpression in Escherichia coli (E. coli) was constructed. For cloningthe protein coding region (nucleotide numbers 11810-12610) of PF0012gene from Pyrococcus furiosus shown in Genbank Acc. NC_003413 usingIn-Fusion (registered trademark) HD Cloning Kit (manufactured by TAKARABIO INC.), two primers PF12F and PF12R were designed. The amino acidsequence of PF0012 is shown in SEQ ID NO:1. The nucleotide sequences ofprimer PF12F and PF12R are shown in SEQ ID NO:3 and SEQ ID NO:4respectively. The genomic DNA from Pyrococcus furiosus as a template,these primers, and PrimeSTAR (registered trademark) HS DNA polymerase(manufactured by TAKARA BIO INC.) were used to amplify the region. Thenucleotide sequence of the amplified region is shown in SEQ ID NO:5. Theamplified fragment was inserted between NdeI and BamHI cleavage sites inplasmid pET15b (manufactured by Merck Millipore Corporation) usingIn-Fusion (registered trademark) HD Cloning Kit. The In-Fusion reactionmixture was used to transform E. coli JM109. From clones exhibitingampicillin resistance, a plasmid for the expression of PF0012, pET-PF12was isolated. From a crude extract of E. coli retaining this plasmid, amismatch endonuclease activity was detected by a method described inExample 2. However, this plasmid was very unstable and caused internaldeletion frequently. Thus the plasmid was not suitable for production ofprotein.

(2) Preparation of pET-optPFl2, Optimized Plasmid for Expression ofPF0012

Since the expression of protein PF0012 using pET-PF12 was not stable, anucleotide sequence was designed to encode the amino acid sequence ofPF0012 by E. coli type codons, and a DNA containing the nucleotidesequence was artificially synthesized. The nucleotide sequence of thisDNA is shown in SEQ ID NO:6. The DNA was digested with restrictionenzymes NdeI and BamHI, and then purified. The DNA fragment thusobtained was inserted between NdeI and BamHI restriction sites in pET15busing DNA Ligation Kit <Mighty Mix> (manufactured by TAKARA BIO INC.).The ligation reaction mixture was used to transform E. coli JM109.Clones exhibiting ampicillin resistance were selected. From the clones,an optimized plasmid for the expression of PF0012, pET-optPF12 wasobtained.

(3) Expression of Protein PF0012

E. coli BL21 DE3 was transformed with plasmid pET-optPF12. Clonesexhibiting ampicillin resistance were isolated. Fresh single colony thusobtained was cultured in 2 ml of an LB medium containing 100 μg/ml ofampicillin (hereinafter, referred to as LB-AP medium) at 37° C.overnight. To 50 ml of an LB-AP medium, 500 μl of the overnight culturewas added, and then cultured at 37° C. for 3 hours. Then, to the culturesolution, IPTG was added at a final concentration of 1 mM, and thencultured at 37° C. for 3 hours. After completion of culture, the culturesolution was centrifuged at 6000×g for 10 minutes to harvest bacteriacells from the culture solution.

(4) Purification of Protein PF0012

The bacteria cells described above were suspended in 3 ml of a solutioncontaining 50 mM Tris-HCl, pH 7.5 and 100 mM NaCl (hereinafter, referredto as Buffer A), and subjected to ultrasonic disruption using VP-5SUltras. Homogenizer (manufactured by TAITEC). After the ultrasonictreatment for 30 seconds was repeated 3 times, the suspension becametransparent.

The suspension after the ultrasonic treatment was heated at 95° C. for10 minutes to denature heat-labile protein, and then centrifuged at17000×g for 10 minutes. A supernatant was collected. To the crudeextract thus obtained, 500 μl of Ni-NTA Agarose (manufactured by QIAGEN)was added, followed by gentle stirring at 4° C. for 2 hours. The resinwas filled into an Econo-Pac (registered trademark) column (manufacturedby Bio-Rad Laboratories, Inc.), and washed with 20 ml of Buffer Acontaining 5 mM imidazole. After washing, 1 ml of Buffer A containing300 mM imidazole was used to elute protein PF0012. An eluate wasdialyzed with Buffer A at 4° C. two times, and then with Buffer Acontaining 50% glycerol at 4° C. overnight to obtain a solutioncontaining protein PF0012.

Preparation Example 3: Preparation of PF0012 Homologous Proteins

(1) Preparation of pET-TBA and pET-MJA, Plasmids for Expression ofPF0012 Homologous Protein

For the purpose of isolating PF0012 homologous proteins from two strainsbelonging to archaebacteria, Thermococcus barophilus andMethanocaldococcus jannaschii, regions encoding the protein were cloned.The amino acid sequences of PF0012 homologous proteins from these twostrains are shown in SEQ ID NO:7 and SEQ ID NO:8 respectively.

From nucleotide sequence information shown in Genbank Acc. CP002372.1and Genbank Acc. NC_000909.1, sequence information (TERMP_01877:nucleotide numbers 1674836-1675591, and MJ_0225: nucleotide numbers215449-216240) corresponding to the PF0012 homologous proteins wereobtained. Primers for cloning the corresponding genes were designed andprepared. For cloning of the TERMP_01877 gene, a pair of primersTBA1877F and TBA1877R were used. For cloning of the MJ_0225 gene, a pairof primers MJ255F and MJ255R were used. The nucleotide sequences ofthese primers are respectively shown in SEQ ID NOs:9, 10, 11 and 12.

The genomic DNA of Thermococcus barophilus or Methanocaldococcusjannaschii as a template, the above-mentioned primer pair, and PrimeSTAR(registered trademark) HS DNA polymerase (manufactured by TAKARA BIOINC.) were used to amplify the region. The nucleotide sequences of theamplified DNAs are shown in SEQ ID NOs:13 and 14. In the same manner asthe preparation of pET-PF12 described above, the amplified DNAs eachwere inserted into plasmid pET15b to obtain plasmid pET-TBA and plasmidpET-MJA.

(2) Expression of PF0012 Homologous Proteins

The Expression and purification of the PF0012 homologous proteins wereperformed in the same manner as the expression of protein PF0012described above, except that pET-TBA and pET-MJA were used as plasmidsand after disruption of bacteria cell, the heat-labile proteins wereprecipitated by high-temperature treatment at 75° C. for 10 minutes.Thus PF0012 homologous proteins, TERMP_01877 and protein MJ_0225, wereobtained.

Example 1: Preparation of Mutant PF0012 Protein

(1) Preparation of pET-optPF12-W77F, Plasmid for Expression of MutantPF0012

A gene encoding a mutant of protein PF0012 in which amino acidtryptophan (codon TGG) at position 77 in protein PF0012 (SEQ ID NO:1)was changed into phenylalanine (codon TTT) was prepared. The tryptophanat position 77 in PF0012 was believed to be an ssDNA binding regionaccording to analysis of homologous protein NucS of protein PF0012 (TheEMBO Journal, 2009, vol. 28, p. 2479-2489). The amino acid sequence ofthe mutant of PF0012 protein (hereinafter, referred to as mutant W77F)is shown in SEQ ID NO:2.

As primers for introduction of the mutation, mutF1 and mutR1 shown inSEQ ID NOs:15 and 16 were prepared. PCR was performed using pET-optPF12as a template, the above-mentioned primers, and PrimeSTAR (registeredtrademark) Mutagenesis Basal Kit (manufactured by TAKARA BIO INC.). Anamplification reaction mixture thus obtained was used to transform E.coli JM109. From clones exhibiting ampicillin resistance, a plasmid forthe expression of mutant W77F, pET-optPF12-W77F was obtained.

(2) Expression and Purification of Mutant W77F

The Expression and purification of mutant W77F were performed in thesame manner as those of wild-type protein PF0012 described above, exceptthat pET-optPF12-W77F was used as a plasmid.

Example 2: Mismatch Cleavage Activity of Protein PF0012

(1) Preparation of Substrates for Measurement of Cleavage Activity—1

To determine the mismatch cleavage activity of protein PF0012,substrates for detection of the activity were prepared.

Two synthetic DNAs, one labeled with FAM at its 5′ end and the otherlabeled with ROX at its 5′ end were mixed and annealed to prepare thesubstrate. Four kinds of the ROX-labeled DNA: ROX-probe-G, ROX-probe-A,ROX-probe-T, and ROX-probe-C were synthesized. The nucleotide sequencesof the four kinds of the ROX-labeled DNA are shown in SEQ ID NOs:17, 18,19 and 20 respectively. The nucleotide sequences of ROX-probe-G,ROX-probe-A, ROX-probe-T, and ROX-probe-C were identical except for the11th base from the 5′ end, wherein the 11th bases are G, A, T, and Crespectively. Four kinds of the FAM-labeled DNA: FAM-probe-G,FAM-probe-A, FAM-probe-T, and FAM-probe-C were synthesized. Thenucleotide sequences of the four kinds of the FAM-labeled DNA are shownin SEQ ID NOs:21, 22, 23 and 24 respectively. The nucleotide sequencesof FAM-probe-G, FAM-probe-A, FAM-probe-T, and FAM-probe-C were identicalexcept for the 15th base (G, A, T, and C respectively) from the 5′ end.From these, one kind of the ROX-labeled DNA and one kind of theFAM-labeled DNA were selected, mixed and hybridized to prepare thesubstrate for detection of the mismatch cleavage activity. The substrateis a double-stranded DNA forming base pairs at positions other than the11th base from the base labeled with ROX and having one mismatched basepair at the position of the 11th base.

(2) Mismatch Cleavage Reaction by Protein PF0012 (Detection by PAGE)

A reaction mixture containing 1 μl of 5 μM one kind of the ROX-labeledDNA, 1 μl of 5 μM one kind of the FAM-labeled DNA, 1 μl of 10×Ex taqBuffer [attached with TaKaRa Ex Taq (registered trademark), manufacturedby TAKARA BIO INC.], protein PF0012, and H₂O was prepared. ProteinPF0012 and H₂O were added so that the total amount of the reactionmixture became 10 μl. After the reaction mixture was incubated at 60° C.for 1 hour, the reaction was stopped. The reaction mixture was subjectedto electrophoresis on 10% polyacrylamide denaturing gel. After theelectrophoresis, fluorescent signals in the gel were detected by FMBIO(registered trademark) (manufactured by Hitachi Solutions, Ltd.).

FIG. 1 a) shows detection of ROX signals. FIG. 1 b) shows detection ofFAM signals from the same electrophoresis gel as the gel for detectionof ROX signals. In the upper parts of the figures, the alphabetsindicated to the left of ROX show the kinds of the used ROX labeled DNA,and specifically, G, A, T, and C show that ROX-probe-G, ROX-probe-A,ROX-probe-T, and ROX-probe-C were used respectively. The alphabetsindicated to the left of FAM show the kinds of the used FAM labeled DNA,and specifically, G, A, T, and C show that FAM-probe-G, FAM-probe-A,FAM-probe-T, and FAM-probe-C were used respectively. The blank showsthat the FAM labeled DNA was not used and only the ROX labeled DNA wasused for the reaction.

In both systems, only when the substrate having a G-G, G-T, T-G or T-Tmismatch was used, a fluorescent signal from a DNA which probablycorresponded to a cleavage fragment was observed in a low molecularregion. This fact shows that protein PF0012 recognizes the positions ofG-G, G-T, T-G and T-T mismatches and has the ability to cleave bothchains of the DNA in the vicinity of the mismatches.

Since the signal from the cleavage fragment did not show a smearpattern, protein PF0012 probably does not have exonuclease activity. Inaddition, when a single fluorescently labelled DNA was used, nofluorescent signal was observed in a low molecular region. Therefore, itwas found that protein PF0012 has almost no activity of cleaving asingle-stranded DNA.

It was found that protein PF0012 has selectivity for the bases ofmismatches which the protein can cleave, high specificity formismatches, and low reactivity to a double-stranded DNA forming normalbase pairs and a single-stranded DNA. The high specificity formismatches of protein PF0012 is advantageous when the protein is addedto a nucleic acid amplification reaction, because the protein is lesslikely to inhibit the reaction by virtue of the high specificity formismatches.

(3) Preparation of Substrates for Measurement of Cleavage Activity—2

To successively observe the mismatch cleavage activity of PF0012,substrates capable of generating a fluorescent signal by being cleavedwere prepared.

DNAs of the nucleotide sequences shown in SEQ ID NOs:25, 26, 27 28 inwhich a quencher eclipse was bound to the 5′ end and FAM was bound tothe 3′ end: DD-probe-G, DD-probe-A, DD-probe-T, and DD-probe-C weresynthesized. As a template DNA for a complementary strand, template-Gand template-T shown in SEQ ID NOs:29 and 30 were prepared. Thefluorescently labeled DNA and the template DNA were mixed to prepare adouble-stranded DNA substrate having a mismatched base pair at theposition of the 5th base from the 5′ end.

(4) Successive Observation of Mismatch Cleavage Reaction by ProteinPF0012

A reaction mixture containing 1.5 μl of 5 μM the fluorescently labeledDNA (any one of DD-probe-G, DD-probe-A, DD-probe-T, and DD-probe-C), 1μl of 25 μM template-G, 2.5 μl of 10×Ex Taq Buffer, protein PF0012, andH₂O was prepared. Protein PF0012 and H₂O were added so that the totalamount of the reaction mixture became 25 μl. The reaction mixture wasincubated at 55° C. for 1 hour, while a change in fluorescent intensitywas observed once every minute. The reaction and fluorescent intensitywere measured by using Thermal Cycler Dice (registered trademark) RealTime System (manufactured by TAKARA BIO INC.).

FIG. 2 shows the fluorescent intensity minute by minute. In thenon-cleaved substrate, the fluorescent from FAM is quenched by eclipse.When the substrate is cleaved by protein PF0012, the distance betweenFAM and eclipse is increased and thus the fluorescent from FAM isobserved.

In fact, only when the substrate containing a G-G or G-T mismatch wasused, an increase in the fluorescent intensity was observed. When thesubstrate containing a G-A mismatch or a G-C base pairing was used, anincrease in the fluorescent intensity was not observed. This experimentshows that protein PF0012 has the activity of cleaving mismatchescomprised of G or T. The cleavage activity on each mismatched base pairis expressed as an initial rate at the time of reaction initiation, thatis, a value calculated from a slope in a period maintaining linearityand correction by the fluorescent intensity of each substrate. When thereaction initial rates calculated form the curves shown in FIG. 2 arecompared, it is found that the cleavage activity of protein PF0012 onG-G mismatches is 2 times higher than that on G-T mismatches.

Example 3: Mismatch Cleavage Activity of PF0012 Homologous Protein

(1) Mismatch Cleavage Reaction by PF0012 Homologous Protein

The mismatch cleavage activity and the base specificity of PF0012homologous proteins from Thermococcus barophilus and Methanocaldococcusjannaschii were examined. The mismatch cleavage activity was observed onthe basis of an increase in the fluorescent intensity as an indicator inthe same manner as the successive observation of mismatch cleavagereaction by protein PF0012 except that the PF0012 homologous protein(protein TERMP_01877 or protein MJ_0225) was used as the enzyme protein.

As a result, as shown in FIG. 3, it is found that the proteins fromThermococcus barophilus and Methanocaldococcus jannaschii have theactivity of cleaving mismatch positions comprised of G or T.

(4) Decrease of Error Rate in PCR Gene Amplification by Addition ofProtein PF0012

Protein PF0012 can recognize a mismatched base pair in a double-strandednucleic acid and cleave both strands of the double-stranded nucleicacid. This protein is heat-resistant and therefore can be added directlyto a high-temperature reaction system such as PCR. The proteinspecifically cleaves double-stranded nucleic acids containing mismatchedbase pairs that are generated during PCR, and thereby occurrence oferrors during the amplification reaction is expected to be inhibited.

(1) PCR with Addition of Protein PF0012

Protein PF0012 was added to a reaction mixture for PCR. The reactionmixture together with a genomic DNA of Thermus thermophilus HB8 as atemplate and four pairs of primers was subjected to PCR. The primerpairs used were Tth1F (SEQ ID NO:31) and Tth1R (SEQ ID NO:32); Tth2F(SEQ ID NO:33) and Tth2R (SEQ ID NO:34); Tth3F (SEQ ID NO:35) and Tth3R(SEQ ID NO:36); and Tth4F (SEQ ID NO:37) and Tth4R (SEQ ID NO:38).Thermus thermophilus HB8 Genomic DNA Solution (manufactured by TAKARABIO INC.) as a template, the above-mentioned primer pairs and proteinPF0012 were added. The PCR was performed using TaKaRa Taq (registeredtrademark) Hot Start Version (manufactured by TAKARA BIO INC.), in 30cycles of 98° C. for 10 seconds, 55° C. for 30 seconds, and 72° C. for 1minute.

(2) Calculation of Error Rate in PCR

DNA fragments amplified as described above were purified by usingNucleoSpin (registered trademark) Gel and PCR Clean-up (manufactured byTAKARA BIO INC.). The DNA fragments were subjected to TA cloning inpMD19 (simple) plasmid (manufactured by TAKARA BIO INC.). Then, 96plasmid clones for each amplified DNA fragment (384 clones in total)were subjected to nucleotide sequencing of the amplified regions.

After sequence information not containing the DNA of interest andsequence information presumed to be an error generated during theprocedure for nucleotide sequencing analysis were removed, thenucleotide sequence of the amplified region was compared with theoriginal genomic DNA sequence to count the number of different bases.The number of different bases was divided by the total number ofanalyzed bases to obtain an error rate.

In the DNA amplified by PCR without protein PF0012, 71 bp of 128826 bpwere errors and the error rate was 0.055%. When PCR was performed withaddition of protein PF0012, 28 bp of 97112 bp were errors and the errorrate was 0.029%.

Thus, errors generated by nucleic acid amplification can be decreased byonly addition of protein PF0012 to a PCR reaction mixture.

Example 5: Mismatch Cleavage Activity of Mutant W77F

Protein PF0012 originally has selectivity for mismatched base pairscomprised of G or T. We prepared an enzyme protein recognizing furtherlimited mismatched base pairs by introducing a mutation into theprotein.

(1) Successive Observation of Mismatch Cleavage Reaction by Mutant W77F

The mismatch cleavage activity and base specificity of mutant W77Fprepared in Example 1 were observed by using the above-mentionedfluorescent DNA substrates. In this experiment, mutant W77F was usedinstead of protein PF0012, and DD-probe-G, DD-probe-T and template-G,template-T were combined. Other reaction conditions were the same asthose in the measurement of successive mismatch cleavage activitydescribed in Example 2(4).

As shown in FIG. 4, mutant W77F cleaved G-G mismatches well, whereas thecleavage activity on G-T mismatches was equal to or less than 1/20 ofthat on G-G mismatches and T-T mismatches were not cleaved. The activityof wild-type protein PF0012 on G-T mismatches was about ½ of that on G-Gmismatches, as shown in FIG. 2. Thus, mutant W77F has more specificcleavage activity on G-G mismatches.

Example 6: Specific Amplification of a Very Small Amount ofContaminating Mutant Gene by Addition of Mutant W77F

(1) Preparation of Template Plasmid

For a mutated gene amplification test, plasmids as a template wereprepared.

From sequence information of a human TP53 gene region shown in GenbankAcc. NG 017013, two primers shown in SEQ ID NOs:39 and 40: TP53CloneFand TP53CloneR were designed and synthesized. The region was amplifiedusing the above-mentioned primers, a human genomic DNA (manufactured byClontech Laboratories, Inc.) as a template, and PrimeSTAR (registeredtrademark) HS DNA polymerase. The nucleotide sequence information of theamplified region is shown in SEQ ID NO:41. The amplified DNA fragmentwas subjected to TA cloning in plasmid pMD19 (simple) (manufactured byTAKARA BIO INC.). A plasmid thus obtained was referred to as pTP53 (G).To change the 99th base G to A in the amplified region of the TP53 geneusing pTP53(G) as a template, TP53AF and TP53AR shown in SEQ ID NO:42and 43 were designed and prepared as primers for introduction ofmutation. The plasmid pTP53(G) as a template, the primer pair forintroduction of mutation, and PrimeSTAR (registered trademark)Mutagenesis Basal Kit were used to prepare mutant plasmid pTP53(A) inwhich G was changed to A at the desired position.

(2) Design and Preparation of Probe for Specific Mutant Gene Cleavage

An oligonucleotide for specific mutant gene cleavage was designed tohave a sequence complementary to a region extending 8 bases in the 5′direction and 7 bases in the 3′ direction from the position on pTP53(G)into which the mutation was introduced, and contain a mutation of C to Gat a position corresponding to the mutation position on pTP53(G). Whenthe oligonucleotide is hybridized with pTP53(G), a G-G mismatch isgenerated. To prevent an extension reaction from the oligonucleotide, ahydroxyl group at the 3′ end of the oligonucleotide is replaced with anamino group. The oligonucleotide is referred to as an oligonucleotidefor specific SNP cleavage, TP53deg. The nucleotide sequence of TP53degis shown in SEQ ID NO:44.

(3) PCR with Suppressed Amplification of Specific Mutant Gene

Plasmid pTP53(G) alone, plasmid pTP53(A) alone, or a mixture of theseplasmids as a template, mutant W77F, and TP53deg were used to performPCR. The mixing ratios of the plasmids were, relative to 100 ng ofpTP53(G), 100 ng (1/1), 10 ng ( 1/10), 1 ng ( 1/100), 100 pg ( 1/1000),and 10 pg ( 1/10000) of pTP53(A). A reaction mixture was preparedaccording to LightCycler (registered trademark) 480 High ResolutionMelting Master (manufactured by Roche Diagnostics). The reaction mixturecontained primers TP53AmpF and TP53AmpR shown in SEQ ID NO:45 and 46respectively at a final concentration of 3 μM as primers foramplification, TP53deg at a final concentration of 3 μM as anoligonucleotide for specific mutant gene cleavage, and mutant W77F.Mutant W77F was added so that the total volume of the reaction mixturebecame 20 μl. PCR and fluorescent detection were performed by usingLC480 (manufactured by Roche). The reaction conditions for PCR comprisedprewarming at 95° C. for 5 minutes, 40 cycles of 3-step reactionconsisting of 95° C. for 10 seconds, 95° C. for seconds and 72° C. for20 seconds, finally as a dissociation reaction 95° C. for 1 minute, 40°C. for 1 minute, and then successive increasing temperature from 50° C.to 95° C., while fluorescent intensity at each temperature was observed.The same measurement was performed using a reaction mixture with noaddition of mutant W77F.

(4) Detection of a Very Small Amount of Contaminating Mutant Gene

FIG. 5 shows amplification curves in the PCR. Table 1 shows Ct valuescalculated from the measurement results. When the reaction mixture didnot contain mutant W77F, little change in the Ct value depending on themixing ratio of the templates was observed. In contrast, when PCR wasperformed with addition of mutant W77F, the reaction using plasmidpTP53(G) alone as the template was inhibited and the Ct value wasincreased by 10 or more. This fact shows that mutant W77F inhibits DNAamplification from the plasmid by 1000 times or more. In the case of theamplification using plasmid pTP53(A) alone as the template, addition ofmutant W77F hardly changed the Ct value, and the amplification washardly inhibited by addition of mutant W77F. When the mixture of theplasmids was used as the template, amplifications depending on theamounts of pTP53(A) were observed. This fact shows that although mutantW77F cleaves a double-stranded nucleic acid containing a G-G mismatchedbase pair formed from pTP53(G) and TP53deg in the PCR reaction mixture,mutant W77F exhibits little cleavage activity on a G-A mismatched basepair formed from pTP53(G) and TP53deg. In addition, it was found thatmutant W77F does not show amplification-inhibiting effect on a nucleicacid containing no mismatch. When the reaction mixture contained 1/10000copies of pTP53(A) relative to pTP53(G), the Ct value was smaller thanpTP53(G) alone. This suggests that the gene region of mutant-type (A)was specifically amplified from the template present in the reactionmixture at the mixing ratio of 1/10000 by virtue of mutant W77F.

TABLE 1 Ct value No addition of mutant Addition of mutant G:A ratio W77FW77F 1:0 22.01 34.55 0:1 18.9 19.79 1:1 18.38 19.79 1:0.1 20.3 23.481:0.01 20.61 27.62 1:0.001 21.38 28.15 1:0.0001 21.22 32.71

The fluorescent intensity of each amplified product was measured whilethe temperature was increased from 50° C. to 95° C. to perform meltingcurve analysis. FIG. 6 shows graphs of first derivation of thefluorescent intensity at each temperature. As shown in FIG. 6 a), whenthe PCR was performed with no addition of mutant W77F, a peak appearedat 90.2° C. in the melting curve analysis of the amplified product usingpTP53(G) alone as the template. When pTP53(A) was used alone as thetemplate, a peak appeared at 89.6° C. In the case of a hetero-doublestrand in which both plasmids were present at the ratio of 1:1, inaddition to the peaks appearing in the case of each plasmid alone, abroad peak appeared at around 88.5° C. Such differences of peaks infirst derivation of melting curves can be utilized to distinguish thetemplate containing wild-type (G) and the template containingmutant-type (A).

In the PCR with addition of mutant W77F, as shown in FIG. 6b ), when themixing ratio of plasmid pTP53(A) was 1/100 or more, the waveform wasalmost the same as that found in the case of using pTP53(A) alone as thetemplate. This fact shows that only the amplification using pTP53(A) asthe template was performed. When the mixing ratio of plasmid pTP53(A)was 1/1000 or 1/10000, the peak at 89.6° C. which probably came frompTP53(A) was observed, and further, the broad peak at around 88.5° C.which probably came from the hetero-double strand was also observed.This fact shows that in these mixing ratios, the fragment from pTP53(A)was preferentially amplified.

In this Example, it was found that the addition of a mutant of proteinPF0012 and an oligodeoxyucleotide which generates a mismatch with thenucleotide sequence of the desired gene region leads to inhibition ofamplification of the gene having the nucleotide sequence, and thus leadsto specific amplification of a gene having a small amount of SNP.

INDUSTRIAL APPLICABILITY

The present invention is useful in broad fields including the fields ofbiotechnology, biology, medicine, and agriculture.

SEQUENCE LISTING FREE TEXT

SEQ ID NO:1; The amino acid sequence of PF0012 from Pyrococcus furiosus

SEQ ID NO:2; The amino acid sequence of PF0012 Y77F mutant

SEQ ID NO:3-4; A designed oligonucleotide primer for PCR

SEQ ID NO:5; The nucleic acid sequence of inserted DNA in the circulardouble stranded DNA for cloning PF0012 gene.

SEQ ID NO:6; The nucleic acid sequence of inserted DNA in the circulardouble stranded DNA for cloning a designed sequence deduced from PF0012amino acid sequence.

SEQ ID NO:7; The amino acid sequence of TERMP_01877 from Thermococcusbarophilus

SEQ ID NO:8; The amino acid sequence of MJ_0225 from Methanocaldococcusjannaschii

SEQ ID NO:9-12; A designed oligonucleotide primer for PCR

SEQ ID NO:13; The nucleic acid sequence of inserted DNA in the circulardouble stranded DNA for cloning TERMP 01877 gene.

SEQ ID NO:14; The nucleic acid sequence of inserted DNA in the circulardouble stranded DNA for cloning MJ_0225 gene.

SEQ ID NO:15-16; A designed oligonucleotide primer for PCR

SEQ ID NO:17-30; A designed oligonucleotide DNA for assay of a mismatchnuclease activity.

SEQ ID NO:31-40; A designed oligonucleotide primer for PCR

SEQ ID NO:41; The nucleic acid sequence of inserted DNA in the circulardouble stranded DNA for cloning partial TP53 gene.

SEQ ID NO:42-43; A designed oligonucleotide primer for PCR

SEQ ID NO:44; A designed oligonucleotide DNA for suppression ofamplification of specific TP53 fragment

SEQ ID NO:45-46; A designed oligonucleotide primer for PCR

The invention claimed is:
 1. A method of inhibiting amplification of anucleic acid having a specific nucleotide sequence in a nucleic acidamplification reaction, the method comprising a step of performing thenucleic acid amplification reaction in the presence of the following (a)to (d): (a) an oligodeoxynucleotide which is designed to generate one toseveral mismatches when the oligodeoxynucleotide is hybridized with thenucleic acid having a specific nucleotide sequence or a complementarystrand thereof; (b) a DNA polymerase; (c) at least one pair ofoligonucleotide primers; and (d) a polypeptide having a mismatchendonuclease activity which is at least one polypeptide selected fromthe group consisting of the following (i) to (vi): (i) a polypeptidehaving an amino acid sequence of SEQ ID NO:1, 7 or 8; (ii) a polypeptidehaving an amino acid sequence which differs from the amino acid sequenceof SEQ ID NO:1, 7 or 8 by substitution, deletion, insertion and/oraddition of 1 to 10 amino acid residues, and having a mismatchendonuclease activity; (iii) a polypeptide having an amino acid sequencewhich shares at least 75% amino acid sequence identity with the aminoacid sequence of SEQ ID NO:1, 7 or 8, and having a mismatch endonucleaseactivity; (iv) a polypeptide having an amino acid sequence of SEQ IDNO:2; (v) a polypeptide having an amino acid sequence which differs fromthe amino acid sequence of SEQ ID NO:2 by substitution, deletion,insertion and/or addition of 1 to 10 amino acid residues other thanphenylalanine at position 77, and having a mismatch endonucleaseactivity; and (vi) a polypeptide having an amino acid sequence whichshares at least 75% amino acid sequence identity with the amino acidsequence of SEQ ID NO:1, in which an amino acid residue corresponding totryptophan at position 77 in the amino acid sequence of SEQ ID NO:1 issubstituted with phenylalanine, and having a mismatch endonucleaseactivity.
 2. The method according to claim 1, wherein the nucleic acidamplification reaction is a polymerase chain reaction (PCR) method or anisothermal nucleic acid amplification method.
 3. A method ofpreferentially amplifying a target DNA, the method comprising inhibitingamplification of a DNA having a nucleotide sequence different from thatof the target DNA in one to several nucleotides by the method accordingto claim
 1. 4. The method according to claim 3, which is used foramplifying a DNA having a wild-type nucleotide sequence or the DNAcontaining a single nucleotide polymorphism mutation, wherein theamplification of the wild-type DNA and the amplification of the DNAcontaining a single nucleotide polymorphism mutation are distinguishedfrom each other.
 5. The method according to claim 4, wherein the singlenucleotide polymorphism mutation correlates with canceration, or atherapeutic effect of an agent for the treatment of a cancer.
 6. Themethod according to claim 3, wherein the DNA amplification reaction isperformed by a polymerase chain reaction (PCR) method or an isothermalnucleic acid amplification method.